Many simple organic molecules including solvents, agrichemicals, drugs and drug candidates become cytotoxic upon biotransformation to reactive electrophilic metabolites. The liver is a major site of foreign compound metabolism and is often a major target for drug and chemical toxicity. Hepatotoxicity remains the single most important factor in the failure of drug candidates late in development (when such failure is extremely expensive in terms of lost investment of resources), and of approved drugs early in open clinical usage (when the cost of failure can also include severe or even fatal injury to patients). Understanding the mechanism(s) by which chemicals cause cytotoxic liver injury remains a major challenge of enormous scientific and practical importance. A major focus of our laboratory has been the identification and characterization of chemically reactive metabolites, the protein adducts they form in living cells, and the specific proteins that become adducted. We have hypothesized that there exists a common subset of cellular proteins whose covalent modification by reactive metabolites initiates cytotoxic events through intracellular signaling mechanisms. Our recent work has identified 28 such candidate proteins. Bioinformatic analysis reveals that 21 of them interact with 165 partner proteins, many of which are members of the MAP kinase signaling pathway. Encouraged by these observations, we propose to test our original hypothesis more thoroughly by (1) expanding significantly the list of known target proteins for chemically-reactive metabolites of several paradigm hepatotoxicants to look for commonality (i.e. functional proteomics); (2) applying bioinformatic methods to analyze target proteins and their first-neighbor interacting partners to uncover common signaling pathways that lead to cytotoxic outcomes; (3) testing the hypothesis that hepatocytes are able to recognize and respond to adducted proteins per se by delivering pre-adducted proteins directly into hepatocytes using protein transduction domains and observing the cells for cytotoxic responses and/or changes in intracellular signaling pathways associated with hepatotoxicity. PUBLIC HEALTH RELEVANCE: Many drugs and chemicals give rise to chemically-reactive metabolites that modify cellular proteins leading to cytotoxicity. We have identified certain proteins whose modification may play a general role in this process, and we now propose to search more broadly for other such proteins and their interacting cellular partners that may form part of a general process underlying reactive metabolite cytotoxicity.